Residence of p450 system enzymes in endoplasmic reticular lipid microdomains

Abstract

P450 is part of an electron transport chain found in the endoplasmic reticulum (ER), with its catalytic function requiring interactions with other ER‐resident enzymes such as NADPH‐cytochrome P450 reductase (CPR). The goal of this study was to examine how the P450 system proteins are organized, and to determine if they are heterogeneously distributed in lipid microdomains (LM). ER from untreated and phenobarbital (PB)‐treated rabbits were partially solubilized with 1% Brij 98 for 5 minutes at 37°C, and LMs were isolated via sucrose gradient centrifugation. Lipid analysis showed that LM fractions were enriched in cholesterol and sphingomyelin, components that are also enriched in LMs found in plasma membrane and are indicative of lipid raft formation. Immune blotting of LM fractions showed that 75% of CYP1A2 and 40% of CPR resided in LM fractions in control ER. In PB microsomes, approximately 94%, 60%, and 65% of CYP1A2, CYP2B4, and CPR, respectively, were found in these regions. Preliminary studies showed that the lipid composition found in LM fractions has a significant effect on substrate metabolism, where the rate of CYP1A2 ethoxyresorufin metabolism was higher in vesicles with the same composition as LM, when compared to simple phosphatidylcholine vesicles. These results suggest that ER‐resident proteins exist in specific lipid microdomains that can influence P450 function. (Supported by NIEHS ES004344)